Magnecytometer for the detection of one or more analytes

ABSTRACT

A system and method for analyzing a sample of liquid having an NMR signal in response to a magnetic field for the presence of an analyte. Included is an NMR device having a testing section that is adapted to contain a liquid and apply a magnetic field to the liquid. A complex comprised of a conjugate having a field gradient bound to the analyte that is of sufficient magnitude to quench the NMR signal of the liquid when in the test section whereby the presence of the complex is determined by the absence of the NMR signal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit U.S. Provisional Application No.61/785,508, filed Mar. 14, 2013 and herein incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

There is a need for efficient, inexpensive diagnostic tools that may beused in the detection of diseases in patients. A cost-effectivediagnostic system that delivers timely results would enhance point ofcare analyses and ultimately save lives.

2. Brief Summary of the Invention

The present invention avoids the general drawbacks of the prior art byusing nuclear magnetic resonance (NMR) spectroscopy that has thesensitivity to detect single magnetic nanoparticles in an aqueoussolution. In one embodiment, the present invention provides a novel NMRmicrocoil spectroscopic flow cytometer (a Magnecytometer), whichperforms ultra-sensitive cell detection and isolation. The inventionuses the technique of binding of antibody-conjugated, super-paramagneticiron oxide nanoparticles (SPIONs) to tumor cells or other cells ofinterest and flow NMR spectroscopy of water in the surrounding buffersolution. The invention utilizes the SPION-induced alteration in the NMRrelaxation of the water in an NMR microcoil as a detection mechanism.The invention has the sensitivity to detect a single cell obtained froma small volume of liquid. The invention may also be applied to thedetection and capture of almost any type of cell, virus ormacromolecule.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic of an embodiment of the invention.

FIG. 2 illustrates a biological complex that may be used with theinvention.

FIG. 3 is a schematic of another embodiment of the invention.

FIG. 4 is a flowchart of an embodiment of the invention.

FIG. 5 is a graph showing volume dependent loading and washing profilesof His-tagged BSA-SPION complex. The first D1 peak shows the amount ofprotein entering the column. The first D2 peak shows protein exiting thecolumn. The second peak of D2 gives the washing profile.

FIGS. 6A and 6B illustrate how SPIONs quench a magnetic signal of aliquid.

FIG. 7 is a graph showing the NMR continuous spectra of BSA complex.Approximately 250 beads were detected.

FIG. 8 is a graph showing the continuous NMR spectrum of completecomplex using BSA.1.6E6 molecules detected.

FIG. 9 is a graph showing the continuous NMR spectrum of a complex withC4-2 cells. Approximately 5 cells were detected.

DETAILED DESCRIPTION OF THE INVENTION

This description is not to be taken a limiting sense, but is made merelyfor the purpose of illustrating the general principles of the invention.The scope of the invention is defined by the appended claims. In apreferred embodiment, the present invention provides a device and methodfor analyzing a sample of liquid having an NMR signal in response to amagnetic field for the presence of an analyte. As shown in FIG. 1, ananalyte 110 such as a single cell, a cancer cell or even a single cancercell, is located in a nuclear magnetic resonance device 100 having asample testing section 102, which may be 1 microliter or less in volume,in which a magnetic field is applied by coil 104 that surrounds tube106. Analyte 110 may be conjugated with a conjugate, which may beparamagnetic or superparamagnetic, having a field gradient that quenchesall or sonic of the NMR signal of the liquid in the sample testingsection 102. Testing is performed by applying a magnetic field when aliquid containing a potential analyte is located in sample testingsection 102 to determine the presence of the analyte 110 based upon theNMR signal 120 of the liquid being quenched by the conjugate.

For detecting a single bead in the device shown in FIG. 1, a bead wasplaced in agarose under high temperature conditions. The temperature wasthen lowered to fix the bead in place inside capillary tube. A coil wasthen wrapped around the capillary and NMR was performed.

To test a small sample size of liquid, another embodiment of theinvention prepares the analyte for analysis by creating a biologicalcomplex 200 as show in FIG. 2. The biological complex is comprised of arecognition ligand 212 bound to the analyte 210. The recognition ligand212 has an affinity to the analyte and an affinity to an affinity columnresulting in the attachment of the biological complex to the affinitycolumn. This permits the conjugate 220 to bind with the analyte and alsocreates an elution solution for analysis when the biological complex isremoved from the column. A biological marker 240 may also be attached tothe analyte 210 for testing of the presence of the analyte prior toattaching the analyte to an affinity column. Moreover, to concentratethe analyte, the presence of the biological marker is also tested as theanalyte passes through the affinity column with the process continuinguntil no analyte is detected passing through the column. After testing,a magnet may be used to remove the biological complexes from the elutionsolution.

For a complex formed in the column using a prostate cancer cell, theNickel agarose bead non-covalently binds the His-Tagged antibody that isattached to a chromophore. The Antibody is then attached to the prostatecancer cell via the receptor PSMA. SPIONs are then attached to the cellvia a streptavidin-biotinylated anti-PSMA 270 and 271.

The above-described embodiment uses a nuclear magnetic resonance (NMR)based flow cytometer (a Magnecytometer) to provide a rapid, specificdetection of small numbers of biological objects, such as cells,proteins, and viruses in native biological fluids without the need forpreprocessing or separations. Since biochemistry is chemistry whichtakes place in water, the invention uses the NMR spectroscopic signalfrom the abundant (˜111 Molar) solvent water protons to produce a largeNMR signal. The detection scheme relies on the modification of a watersignal in the presence of a conjugate that acts as a signal modifier,such as super-paramagnetic iron oxide nanoparticles (SPIONs). SPIONspossess no intrinsic magnetic field, but when placed into the staticfield of an NMR magnet, they become magnetically-saturated and producemagnetic field gradients which extend radially up to 50 micrometers.Water protons diffusing in these strong gradients experience decreasedtransverse relaxation times with subsequent line broadening. Using thisproperty of SPIONs it can be shown that a single 1 micrometer SPIONwould perturb the NMR signal from ˜0.5 nL of the surrounding watercontaining an easily-detectable ˜10¹⁷ protons.

Accordingly, by matching the volume of liquid in test section 102 with aconjugate having a field gradient that quenches the NMR signal of theliquid in the test section 102 a single analyte may be detected. In aspecific embodiment, matching an 80 micrometer diameter NMR microcoilhaving a testing section containing this volume of water with a singleSPION conjugate will quench the NMR signal from the water in thisvolume. SPIONs can also be conjugated to molecules, such as antibodies,that recognize other biological molecules. Therefore, attaching aconjugated SPIONs to biological analytes, and separate analyte-boundSPIONs from those lacking bound analytes, the microcoil NMR 100 may beused to detect conjugates, such as SPIONs, acting as surrogates, orsignal amplifiers, for cells, viruses or molecules that are associatedwith a variety of diseases. Signal modifiers such as SPIONs may beattached as conjugates to antibodies directed against cell receptors andthen may be used for single cell detection.

Another aspect of the invention is a method for ensuring that only thoseconjugates that have bound to an analyte pass through the NMR microcoilfor counting. Another aspect of the invention addresses the fact thatbiological objects of interest are often dilute, so that large fluidvolumes would need to be processed or examined. The passage of largevolumes (1-100 mL) of fluid through an NMR microcoil would requireinconveniently-large amounts of time.

The present invention addresses both these considerations with the aidof affinity column chromatography as shown in FIG. 3 which allows abiological complex 300, as described above, to be assembled in astep-wise fashion on column 301, by washing off the unbound, andnon-specific material 310, and only eluting the conjugate or SPION-boundcomplex 300 that is passed through the NMR device 100.

During the assembly of the complex, the analyte may be concentrated byprocessing arbitrary amounts of fluid. The analyte may also be recoveredwith a magnet after it has passed through the NMR microcoil for lateruse because the analyte would be now attached to magnetic beads 220. Inthis manner it is possible to use antibodies 212 as recognition ligandsfor single cell detection even in large amounts of biological fluids.

Possible uses for the invention include, but are not limited to,measuring circulating tumor cells in blood. The large number of unwantederythrocytes and other blood cells would constitute a non-interferingbackground. It is also important to note than magnecytometry isminimally invasive, requiring only a small amount of blood ofapproximately 30 microliters. Thus, the invention may improve the waydoctors diagnose diseases such as prostate cancer which oftenmetastasizes and therefore circulating tumor cells can be found in theblood, even in early stages of the disease.

In one preferred embodiment, the affinity column 301 may be packed withnickel (Ni)-agarose 303, then a His-tagged antibody 212 is run throughthe system and recycled to fully saturate the nickel agarose column. Thenext step is to run a sample through the column and wash with a 2.5 mMimidazole (Sigma™, St. Louis, Mo.) wash buffer, this ensures there areno containments other than the analyte 210 of interest attached to thecolumn that will decrease the chance of a false positive running throughthe NMR.

As shown in FIG. 4, two spectrophotometers 402 and 406 on either side ofthe column may also be used. The first detector 402 detects proteinsgoing into column 301. The second detector 406 detects for a biologicalmarker such as a chromophore 240 that is attached to the His-taggedantibody 212. The His-tagged antibody attaches to the Ni-agarose columnfor the direct quantification of His-Tagged antibody that elutes fromthe column that is then released by the imidazole competitively bindingto the Ni-agarose. The conjugates 220, which may be SPIONs, are then runthrough the column which bind via a streptavidin 250 conjugated SPIONsand a biotinylated antibody 252 that then attaches to cells of interestcompleting biological complex 200 as shown in FIG. 2.

Once bound, the sample is eluted with a 200 mM imidazole elution bufferto cleave the His-tagged antibodies off the column that then run throughthe NMR. The properties of SPION may then be used to detect for theanalytes or cells of interest. Samples from the NMR may also be gatheredin a fraction collector for further analysis such as determination ofthe protein concentration, and iron assays by using a magnet.

The NMR was Bruker™ (Madison, Wis.) MiniSpec and the solenoid coil wasdeveloped using 50 gauge copper wire wound around the outer diameter ofa glass capillary tube 106 (O.D. 170 μm; I.D. 100 μm). The π/2 pulselength for this coil is 80 μs. The resonance frequency is 40.015 MHz,and contains a 100 cm permanent magnet (MRT Inc. Tsukuba Japan), alldata was collected using a Hewitt Packard Windows XP™ Running Magritek(Welington, New Zealand) Prospa™, and WinDaq™ (collects optical datafrom detectors) on one hard drive. Data was collected via a Magritek Keaand then stored to the console. A continuous pulse repeat macro waswritten with the following stipulations: The macro repeats a RF pulseevery 0.861 seconds for a continuous reading on each coil volume. Amacro was also written to integrate the FID data for every RF pulse andgenerate a plot.

The nickel agarose was obtained from Thermo Scientific™ and contains 6%beaded agarose with a binding capacity of 10 mg/mL. Anti-PSMA, cloneJ591 antibody was purchased from Neil H. Bander, MD (Cornell College ofMedicine, USA). Proteins were His-Tagged using the Solulink™ procedureand reagents. The J-591 were biotinylated using the Lightning Link™Biotin conjugation kit Type A from Innova Bioscience. The SPIONs aremanufactured by MagSense™ and are streptavidin modified. The wereconjugated with biotinylated anti-PSMA antibody J591 at a ratio of75×10³ beads per ng antibody; Typically 200 ng J591 antibody wereincubated in 0.5 mL PBS (Phosphate Buffered Saline) containing 15×10⁶beads by gentle end-over-end rotation at room temperature.Antibody-functionalized SPIONs were then combined with prostate tumorcells in PBS at a ratio of 300 beads per cell.

For an embodiment in which prostrate cancer cells were detected usingthe invention, the PSMA receptor 260 was selected as a preferentialtarget for SPION labeling and attaching the His-tags. The antibody forPSMA and the protein complex BSA had a His-tag placed upon them alongwith the hydrazonechromophore following the manufacturer's (Solulink™)procedure: The protein must first be desalted using Zeba (ThermoScientific™, Rockford, Ill.) desalt columns. Protein concentrationdetermination was also performed using Pierce™ Micro HCA Kit. S-4FB(linker containing chromophore) was subsequently added, using 2 moleequivalents of S-4FB per protein. The protein then was desalted usingZeba desalting columns, and protein concentration was then determined.The molar substitution ratio was subsequently determined based on theamount of protein. Protein conjugated to S-4FB was then conjugated to 6×His-Tag and was incubated at room temperature for 16 hours. Removal ofexcess His-tags was performed using MicroconUltracel YM-3 spin columnwith a 3000 molecular weight cut-off. The biological complex was thenwashed twice with PBS and concentration of protein complex determined,and labeling was determined using absorbance at 360 nm. The antibodieswere also conjugated to biotin, using the Lightning Link™ Biotinconjugation Kit from Innova Biosciences. Protein concentrationdetermination was done prior to biotinylation, then 1 μL of LL-modifierreagent was added for every 10 μL of antibody used. This mixture wasthen added to Lightning Link™ mix and resuspended resulting in a mixturethat was then incubated for 3 hours at room temperature and 1 μL ofLL-quencher was added for every 10 μL of antibody used. Proteinconcentration was again determined.

The C4-2 prostate cancer cell line that was grown in vented cell cultureflasks (BD Biosciences, San Jose, Calif.) in Dulbecco's modified Eagle'smedia (DMEM) containing: 4.5 g/L glucose and 2 mM L-glutamine (Sigma™,St. Louis, Mo.), and supplemented with 10% fetal bovine serum (FBS;Hyclone, Logan Utah) and penicillin/streptomycin at 100 U (Sigma™). Thecells were grown at 37° C. in a humidified 95% O₂/5% CO₂ atmosphere topassage numbers typically not exceeding 35 to avoid genotypic drifts;Cell detachment was in 0.5% trypsin containing 0.02% EDTA (Sigma™) for30 seconds upon reaching 60-80% confluence; Trypsin action was stoppedby adding medium and cells were harvested by centrifugation at 150×g for10 minutes at room temperature, then resuspended in phosphate bufferedsaline (PBS; Sigma™). Cell numbers were counted using a hemocytometer.

FIG. 4 illustrates an exemplary process flow of an embodiment of theinvention. As shown, sample 400 is injected and data is acquired bydetector 402, which may be set at 280 nm or any other suitablewavelength, and stored by a processor. The sample then flows throughaffinity column 404 where a desired protein binds to the His-taggedantibody specific for the cell or protein of interest and the remainderof the sample flows past detector 406 and can be recycled through thecolumn to ensure maximum binding. Then the wash buffer containing 2.5 mMimidazole flows through the system releasing excess particles andmolecules not bound to column. Elution buffer containing a highconcentration (200 mM) of imidazole then releases His-Tagged antibodiesbound to the column, also releasing cells/proteins of interest bound toSPIONs.

Detector 406 may be set at 360 nm, or any other suitable wavelength,detects the chromophore 240 attached to His-Tagged antibodies 212 thatrun through the NMR. Small quantities of SPIONs 220 can therefore bedetected. The embodiment may be used to process small quantities ofcells labeled with magnetic beads 220 (˜5 cells) for detection.

Continuing to pass analytes past detector 406 until no reading or asufficiently low reading is obtained. This reduces false positives fromoccurring as shown in FIG. 5. A spectrophotometer functions as adetector was placed before the affinity column to detects the amounts ofprotein prior to entering the column as described above. For an exampleused in accordance with the present invention, FIG. 5 shows that 368.11mg/mL of protein entered the column as shown by peak 510 for detector402 and 56.458 mg/mL of His-Tagged protein exited the column asdemonstrated by peak 512 for detector 406. Peak 510 shows proteinentering the column. Peak 512 shows protein exiting the column. Peak 514shows the wash buffer releasing unbound SPIONs and gives the washingprofile.

As further shown in FIG. 1, a single biological complex 200 identifiedas number 110 was placed in test chamber or section 102 of device 100.NMR was then performed showing the quenched water proton signal 120indicating the presence of the analyte of interest. The detection of asingle bead in the test chamber may then be used to count the number ofanalytes present as an elution solution flows through the device.

FIGS. 6A and 6B illustrate the effect on water protons when stimulatedby SPIONs at varying concentration of magnetic beads. FIG. 6A showscells that do not express PSMA and therefore are not magneticallylabeled with SPIONs. FIG. 6B shows cells that overexpress PSMA and bindSPIONs.

FIG. 7 illustrates the continuous NMR spectrum of the water protonsbeing quenched by the BSA complex. An after iron assay was performed andit was determined that 3.6E6 beads and 1.8E6 BSA molecules (BSA bindstwo beads per molecule) were bound and eluted from the column. FIGS. 8and 9 are the NMR continuous spectra of C4-2 prostate cancer cells,which over express PSMA. The data shows that the cells were successfullybound to the column and detected using NMR. It was determined thatapproximately 5 cells were detected and characterized. Portion 800 showsthe His-Tagged attached to the protein of interest. Portion 802 showsthe wash removing the unbound SPIONs. Portion 902 shows C4-2 cells.Portion 900 shows the wash removing the unbound SPIONs.

What is claimed is:
 1. A method for analyzing a sample of liquid havingan NMR signal in response to a magnetic field for the presence of asingle analyte, the method comprising the steps of: conjugating saidsingle analyte with a conjugate, said conjugate having a field gradientthat quenches the NMR signal of the liquid; placing said single analytein a sample testing section of a nuclear magnetic resonance device, saidsample test section sized to receive a single analyte and to contain avolume of liquid such that said field gradient of said conjugatequenches the NMR signal of the liquid in said sample testing section;applying a magnetic field to said single analyte when said singleanalyte is located in said sample testing section; and determining thepresence of said single analyte based upon said conjugate quenching theNMR signal of the liquid in said sample testing section.
 2. The methodof claim 1 wherein said analyte is a single cell.
 3. The method of claim1 wherein said analyte is a single cancer cell.
 4. The method of claim 1wherein said conjugate is paramagnetic or superparamagnetic.
 5. Themethod of claim 1 wherein said sample testing section is sized tocontain a volume of liquid such that said field gradient of saidconjugate of said single analyte quenches the entire NMR signal of theliquid in said sample testing section.
 6. The method of claim 1 whereinsaid sample test section has a volume of 1 microliter or less.
 7. Themethod of claim 1 further comprising the steps of preparing said analytefor analysis comprising the steps of: creating a biological complexcomprised of a recognition ligand bound to said analyte by providing arecognition ligand that has an affinity to said analyte and an affinitycolumn; attaching said biological complex to said affinity column; andconjugating said conjugate to said analyte and creating an elutionsolution for analysis by removing said biological complex from saidcolumn.
 8. The method of claim 7 further comprising the steps of:attaching a biological marker to said analyte; testing for saidbiological marker prior to attaching to said analyte to said affinitycolumn; testing for said biological marker after said analyte passesthrough said affinity column; and continuing to pass said analytethrough said column until no analyte is detected passing through thecolumn.
 9. A method for using a nuclear magnetic resonance device tocount one or more analytes one-at-a-time in a sample liquid having anNMR signal in response to a magnetic field comprising the steps of:attaching a biological marker to said analytes; testing for saidbiological marker prior to attaching said analytes to an affinitycolumn; testing for said biological marker after said one or moreanalytes pass through said affinity column; continuing to pass said oneor more analytes through said column until no analytes are detectedpassing through the column to create an elution solution; said elutionsolution containing one or more biological complexes comprised ofanalytes bound to recognition ligands, said biological complexes formedby conjugating a conjugate to said one or more analytes, said conjugatehaving a field gradient that quenches the NMR signal of the liquid;attaching said one or more biological complexes to said affinity column;passing said elution solution through a nuclear magnetic resonancedevice having a sample testing section in which a magnetic field isapplied, said sample test section is sized to receive a singlebiological complex and to contain a volume of liquid such that saidfield gradient of said conjugate of said single biological complexquenches the entire NMR signal of the liquid in said sample testingsection; applying a magnetic field to said single biological complexwhen said biological complex is located in said sample testing section;and counting said biological complexes one-at-a-time by determining thepresence of a biological complex based upon said NMR signal from theliquid being entirely quenched by said conjugate.
 10. The method ofclaim 9 wherein a magnet is used to remove said biological complexesfrom said elution solution.
 11. The method of claim 9 wherein saidanalyte is a single cancer cell.
 12. The method of claim 9 wherein saidconjugate is paramagnetic or superparamagnetic.
 13. The method of claim9 wherein said sample test section has a volume of 1 microliter or less.14. The method of claim 3 wherein said analyte is a single pancreaticcancer cell.
 15. The method of claim 11 wherein said analyte is a singlepancreatic cancer cell.
 16. A method for analyzing a sample of liquidhaving an NMR signal in response to a magnetic field for the presence ofan analyte, the method comprising the steps of: conjugating said analytewith a conjugate, said conjugate having a field gradient that quenchesthe NMR signal of the liquid; placing said analyte in a sample testingsection of a nuclear magnetic resonance device, said sample test sectionis sized to contain a volume of liquid such that said field gradient ofsaid conjugate quenches the entire NMR signal of the liquid in saidsample testing section; applying a magnetic field to said analyte whensaid analyte is located in said sample testing section; and determiningthe presence of said analyte based upon said conjugate quenching theentire NMR signal of the liquid in said sample testing section.
 17. Themethod of claim 16 wherein said analyte is a single cell.
 18. The methodof claim 1 wherein said analyte is a single cancer cell.
 19. The methodof claim 16 wherein said sample testing section has a volume of 1microliter or less.
 20. The method of claim 16 wherein said sampletesting section is sized to only receive a single analyte.